Hyperbaric oxygen therapy compared to pharmacological intervention in fibromyalgia patients following traumatic brain injury: A randomized, controlled trial

Fibromyalgia is a chronic pain syndrome with unsatisfactory response to current treatments. Physical trauma, including traumatic brain Injury (TBI) is among the etiological triggers. Hyperbaric Oxygen therapy (HBOT) is an intervention that combines 100% oxygen with elevated atmospheric pressure. HBOT has been applied as a neuro-modulatory treatment in central nervous system–related conditions. The current study investigated the utility of HBOT for TBI–related fibromyalgia. Fibromyalgia patients with a history of TBI were randomized to either HBOT or pharmacological intervention. HBOT protocol comprised 60 daily sessions, breathing 100% oxygen by mask at 2 absolute atmospheres (ATA) for 90 minutes. Pharmacological treatment included Pregabalin or Duloxetine. The primary outcome was subjective pain intensity on visual analogue scale (VAS); Secondary endpoints included questionnaires assessing fibromyalgia symptoms as well as Tc-99m-ECD SPECT brain imaging. Pain threshold and conditioned pain modulation (CPM) were also assessed. Results demonstrated a significant group-by-time interaction in pain intensity post-HBOT compared to the medication group (p = 0.001), with a large net effect size (d = -0.95) in pain intensity reduction following HBOT compared to medications. Fibromyalgia related symptoms and pain questionnaires demonstrated significant improvements induced by HBOT as well as improvements in quality of life and increase in pain thresholds and CPM. SPECT demonstrated significant group-by-time interactions between HBOT and medication groups in the left frontal and the right temporal cortex. In conclusion, HBOT can improve pain symptoms, quality of life, emotional and social function of patients suffering from FMS triggered by TBI. The beneficial clinical effect is correlated with increased brain activity in frontal and parietal regions, associated with executive function and emotional processing.


FMS definition and etiology
FMS is a condition characterized by chronic widespread pain and diffuse tenderness, along with symptoms of fatigue, non-restorative sleep and cognitive difficulties [1,2].
Over recent years, significant progress has been made regarding the pathogenesis of FMS. Currently, FMS is considered to represent a prototype of central sensitization, i.e. a condition characterized by an increase in the transmission and processing of pain within the central nervous system [3,4]. The concept of central sensitization has subsequently been applied to a spectrum of overlapping "functional" conditions (e.g. Irritable Bowel Disorder -IBS, Temporomandibular Joint Disorder -TMJD etc), and has been supported and upheld by converging lines of evidence. On the one hand, psychophysical pain testing has clearly demonstrated phenomena such as a decrease in CPM (previously referred to as Diffuse Noxious Inhibitory Control, or DNIC) in many FMS patients [5], indicating a reduced capacity of the CNS to inhibit pain in these individuals. On the other hand, starting over a decade ago with the findings of Graceley et al [6], fMRI has been utilized in order to demonstrate the increased response of pain processing areas within the brain to experimental pain stimulation, among FMS patients. More recent functional imaging paradigms have uncovered additional layers of information regarding the nature of CNS central sensitization in FMS, particularly regarding the phenomenon of increased connectivity between particular brain areas in these individuals. Thus, Napadow et al have described an increase in connectivity between the DMN and the insula in FMS [7] as well as an association between decreased connectivity and reduced clinical pain [8]. Recent evidence has hinted at the possibility, that this increased connectivity may be modified by treatment modalities which bring about a clinical improvement in FMS, both pharmacological [9] and nonpharmacological [10]. Thus, neuro-plasticity appears to be a feasible concept in FMS, with direct relevance for the lynchpin of the FMS pathogenesisthe central sensitization paradigm. This understanding opens the window to a variety of possible interventions aimed at achieving the neuro-plasticity goal.

HBOT
HBOT is a well-known and reliable therapeutic modality, which has been implemented in a variety of clinical conditions. HBOT, the application of hyperbaric pressure in conjunction with increased oxygen content, has been shown in several clinical studies to have the capacity to induce neuroplasticity in injured brains even years after an acute insult [11][12][13][14][15][16][17]. HBOT induces neuroplasticity by stimulating cell proliferation [18], promotes neurogenesis of endogenous neural stem cells [19], regenerates axonal white matter [20], improves maturation and myelination of injured peripheral and cranial neural fibers [21,22], induces brain angiogenesis [12], and stimulates axonal growth thus increasing the ability of neurons [23,24]. To date, two prospective randomized controlled trials have demonstrated the efficacy of HBOT in fibromyalgia [25,26]. The improvement was demonstrated in all aspects of FMS including pain threshold, fatigue, distress and quality of life [25,26].
A growing amount of evidence has been accumulating over recent years regarding the efficacy of HBOT in the treatment of PPCS such as those caused by mTBI [27], which is defined as alteration of brain function caused by external forces, with one or more of the following: loss of consciousness for a duration of 0-30 minutes, posttraumatic amnesia for a duration of less than 24 hours, and Glasgow Coma Scale grade of 13-15 [28]. Specifically, HBOT appears to be capable of inducing cerebral angiogenesis, thus improving perfusion to chronically damaged brain tissue even months to years after the injury [29]. Thus, as recently reviewed by Figueroa et al, 5 out of 5 peer-reviewed clinical trials have demonstrated therapeutic effects of HBOT on symptoms of mTBI/PPCS as well as PTSD [30].
One possible mechanism explaining the neuro-plasticity potential of HBOT relates to the effect on the mitochondrial function of Glia cells [31], a cell population which is postulated to play an integral role in the pathogenesis of central sensitization and chronic pain [32,33]. Breathing oxygen under hyperbaric conditions has the capacity to significantly increase brain oxygen tension, increasing tissue oxygenation and oxygen delivery into Glial mitochondria [34,35]. The etiology of FMS is considered to be multifactorial. A genetic underpinning is widely assumed [36] but is most probably polygenic, similar to other complex human disorders. In addition, various triggers have been associated with the fibromyalgia syndrome, including (but not limited to) physical trauma [37], such as whiplash injury [38] and head injury, infection (e.g. EBV) [39] as well as stress, both acute and chronic.
Notably, in some cases a specific trigger such as a motor vehicle accident can easily be elicited in the history as occurring in close proximity to the development of symptoms, while in other cases no such specific event can be identified.
We have previously studied the efficacy of HBOT as a treatment for FMS in a prospective, active control, crossover clinical trial [25].

Study Objectives
In the proposed study, we intend to both repeat and expand our previous findings, treating FMS patients with HBOT while performing an extensive batter of evaluation both before and after treatment. Our goal is to both establish the role of HBOT in the treatment of FMS as well as to study in depth the mechanisms of neuroplasticity involved and the effect of HBOT. Through extensive advanced imaging and laboratory investigation we also hope to shed significant additional light on the underlying pathogenesis of the fibromyalgia syndrome.
In the current study, we plan to compare HBOT to current standard of care of FMS (pharmacological and nonpharmacological). Notably, since HBOT is associated with a significant investment of time and resources (on behalf of both the patient as well as the health systems), one would need to demonstrate a decisive advantage of this method over the currently recommended standard of care in order to justify the associated investment.
In view of the above mentioned data regarding the efficacy of HBOT in the treatment of mTBI, we would like to focus our study on cases of FMS in which a clear physically traumatic trigger is evident.

Study population
The study will include 70 fibromyalgia patients in whom physical trauma, such mTBI, could be considered as the trigger for FMS. Each participant will be examined at the time of recruitment and a diagnosis of FMS will be verified, based on the updated 2016 diagnostic criteria [40]. These criteria are based on the use of the widespread pain index (WPI) and the Symptom severity Score (SSS).
Exclusion criteria will include the presence of systemic inflammatory disorders including inflammatory rheumatological and autoimmune disorders. Patients suffering from active malignancy, chronic ongoing infection and major psychiatric disorders (excluding anxiety) will be excluded. Patients currently or previously treated with Duloxetine (Cymbalta) or Pregabalin (Lyrica) will also be excluded.
In the current study we will recruit patients not currently being treated with medications specific for FMS, including SSRI, SNRI, gabapentanoids and tricyclics, opiods and medical cannabis. Patients who are on such treatment will be required to discontinue treatment 2 weeks before recruitment.
In addition, patients who have one of the following criteria will also be excluded: Patients will be excluded if they will have one of the following criteria: had been treated with HBOT for any other reason prior to their inclusion; Chest pathology incompatible with pressure changes (including active asthma); Inner ear disease; Claustrophobia; Inability to perform awake brain MRI test; Previous neurologic conditions (eg.

Study protocol
Patients will undergo randomization upon recruitment to one of the two study groups.
One group will proceed to a course of HBOT treatment while the second group will commence with standard treatment for FMS, as outlined in the Israeli guidelines for the diagnosis and treatment of FMS [41]. These patients will be given detailed education regarding the nature of FMS as well as recommendations regarding nonpharmacological interventions recommended for FMS, including graded physical exercise, hydrotherapy, movement-meditative treatments (e.g. Tai Chi) and cognitive behavioral treatment (CBT).

HBOT protocol
A total of 60 daily hyperbaric oxygen treatment sessions will be administrated 5 days per week. Each session will include exposure of 90 minutes to 100% at 2 ATA, with 5 minutes air breaks every 20 minutes.

Pharmaceutical protocol
Patients will be offered pharmacological treatment with one of the two medications currently licensed for the treatment of FMS in Israel, i.e. Cymbalta and Lyrica.
Treatment with Lyrica will start at a dose of 75 mg at bedtime while treatment with Cymbalta will start at a dose of 30 mg a day (in the morning). After a period of 6 weeks patients will be evaluated and dose will be adjusted as necessary. Patients may also be switched from one medication to the other based according to clinical judgment.

Crossover
After 3 months of either pharmaceutical or HBOT, once the 2nd evaluation is completed, all patients in both groups will be offered to switch to the alternative treatment group.

Primary endpoint
The primary end point of the study will be the measurement of daily pain on a (0-10) Visual analogue Scale (VAS) [42]

Secondary endpoint
The secondary end-points of the study will include the following:

Fibromyalgia syndrome symptoms and quality of life questioners
• Global Pain Scale (GPS) [43].

Cognitive functions
Cognitive functions will be evaluated by 2 test batteries: Mindstreems and CATAB.

Mindstreems cognitive battery test
The Mindstreams battery includes several cognitive tests devised to check various aspects of brain capabilities. In the current study we will evaluate the cognitive indices based on the scores of the 6 cognitive tests listed below, which are expected to be relevant for mTBI. For detailed description of all cognitive tests in Mindstreams battery see [50]. The tests are: • Verbal memory: Ten pairs of words are presented, followed by a recognition test in which the first word of a previously presented pair appears together with a list of four words from which the patients choose the other member of the pair. There are four immediate repetitions and one delayed repetition after 10 min.
• Non-verbal memory. Eight pictures of simple geometric objects are presented, followed by a recognition test in which four versions of each object are presented, each oriented in a different direction. There are four immediate repetitions and one delayed repetition after 10 min.
• Go-No-Go test. In this continuous performance test, a colored square (red, green, white or blue) appears randomly on the center of the screen. The patient in then asked to respond quickly only for red squares by pressing the mouse button, and inhibit his reaction to any other colored square.
• Stroop test. Timed test of response inhibition modified from the Stroop paper-based test. In the first phase, patients choose a colored square matching the color of a general word (for example, the word "Cat" appears in red letters, the patient must choose the red square out of two colored squares in the following screen). In the next phase (termed the Choice Reaction Time test), the task is to choose the colored square matching the name of the color presented in white letter-color. In the final (Stroop interference) phase, patients are asked to choose the colored square matching the color and not the meaning of a former color-naming word, presented in an incongruent color (for example, the word "RED" appears in green letters, the patient is asked to choose the color green and not red, a task requiring the ability to inhibit an automatic response to the meaning of the word).
• Staged information processing test. Timed test requiring a reaction based on solving simple arithmetic problems (pressing right/left mouse button if the answer higher/lower than 4, respectively) with three levels of information processing load (single digit, two digits addition/subtraction and three digits addition/ subtraction problems), each containing three speed levels (3, 2, and 1 second for the presentation of the stimuli). object on a computer screen by moving a paddle horizontally so that it can "catch" the falling object.
To assign scores, Mindstreams data will be uploaded to the NeuroTrax central server.
Outcome parameters will be calculated using custom software blind to diagnosis or testing site. To minimize differences related to age and education, each outcome parameter will be normalized and fit to an IQ-like scale (mean=100, STD=15) according to patient's age and education. We note that the score evaluation will be based on normative data from cognitively healthy individuals collected in controlled research studies that were part of more than 10 clinical sites [51].

CANTAB cognitive battery test
Patients' cognitive functions will be assessed by CANTAB computerized cognitive tests (Cambrdige cognition , England) [52]. attention, decision making, social cognition [53,54]. A global cognitive score will be computed as the average of all index scores for each individual. Notably, the patients will be given three different test versions of the CANTAB test battery at baseline, 3 months control period and after HBOT, to allow repeated administrations with minimal learning effect. Test-retest reliability for those versions will be evaluated and found high, with no significant learning effect [55].

Brain imaging
Brain imaging will include 2 type of imaging: perfusion MRI+DTI including resting state functional MRI and Brain SPECT.

Perfusion MRI+DTI
The MRI protocol includes the following sequences: Axial T1, T2, FLAIR, Axial  • Resting state fMRI (rsfMRI\ R-fMRI): a method of functional brain imaging that can be used to evaluate regional interactions that occur when a subject is not performing an explicit task. This resting brain activity is observed through changes in blood flow in the brain which creates what is referred to as a blood-oxygen-level dependent (BOLD) signal that can be measured using fMRI.

Brain SPECT
Brain single photon emission computed tomography (SPECT) will be conducted using Both pretreatment and post-treatment SPECT images will be normalized to the median maximal brain activity in the entire brain and will be then reoriented into Talairach space using Oasis Neurology -NeuroGam application (Segami Corporation, Columbia, MD, USA) to identify Brodmann cortical areas and to compute the mean perfusion in each Brodmann area (BA).

Brain Network Activation (BNA™) Analysis
BNA™ Analysis System is a software only device that utilizes advanced algorithms to analyze the brain network activity of the brain from the recorded EEG data (AMAR: 27860001). The BNA™ Analysis system provides both qualitative network maps of the activity as well as quantitative scores that can be used as a neuromarker for disease.
Event Related Potentials (ERPs), which are temporal reflections of the neural mass electrical activity of cells in specific regions of the brain that occur in response to stimuli, may offer such a method, as they provide both a noninvasive and portable measure of brain function. The ERPs provide excellent temporal information, but spatial resolution for ERPs has traditionally been limited. However, by using highdensity electroencephalograph (EEG) recording spatial resolution for ERPs is improved significantly [56][57][58][59]. The paradigm for the current study will combine neurophysiological knowledge with mathematical signal processing and pattern recognition methods (BNA™) to temporally and spatially map brain function, connectivity and synchronization.
The following parameters will be evaluate by BNA™ technology: • Auditory Oddball -The auditory oddball task is a classic EEG paradigm that has been studied extensively and used in a variety of neurological patient populations.
The task involves executive functions, attention and memory processes. In this task, subjects hear a series of auditory tones and are required to react according to the tones. Tones are presented once every 1.5 seconds on average. A total of 80% of the sounds ("Sensory") are 2000 Hz tones. A total of 10% of the sounds ("Memory") are 1000 Hz tones to which participants respond by pressing a button. The remaining 10% of sounds ("Attention") are complex sounds.
• trial began with the thermode applied to the skin, followed by the triggering of the heat stimulus, and ended with a beep, which prompted subjects' to rate the pain produced by the heat stimulus. This thermode application was repeated in a clockwise manner on the designated area of the forearm. On Visit 1, subjects underwent a temperature-determination protocol, in which they received three different heat stimuli of the same temperature, ranging from 38°C to 52°C. Subjects

Pain threshold and conditioned pain modulation (CPM)
Pressure pain threshold (PPT) will be assessed using a handheld computerized pressure algometer with a circular 1 cm2 probe (AlgoMed, Medoc LTD, Israel). PPTs will be measured three times at the upper trapezius muscle. The baseline pressure applied will be 0 kPa, with incremental increases of 30 kPa per second, up to a maximal pressure of During the pain intensity evaluation (CPM efficacy) brain cortical activity will be continuously monitored using the EEG -DELPhI system (Amar: 570406) evaluating the cortical manifestation of the chronic pain and its correlation with the evaluated CPM efficiency.

Pain evaluations
Participants evaluated pain intensity with a computerized numeric pain rating scale (CNPRS) ranging from 0 (no pain) to 10 (most intense pain tolerable).
• Heat pain threshold and heat pain tolerance -Heat pain threshold is determined as the minimum temperature causing pain and heat pain tolerance is established as the highest tolerable temperature.
Thermal pain is induced with thermal electrode (thermode). Thermode temperature will initially be set at 32.0°C and gradually increase at a rate of 0.3°C/sec.
Participants will be instructed to report when the sensation produced by the thermode changed from heat sensation to pain (heat pain threshold) and when the pain became unbearable (heat pain tolerance). This procedure will be conducted twice for every subject and the mean of the two trials will be calculated. The thermode will be placed on adjacent areas of the forearm for every trial to avoid primary skin hyperalgesia.
• Heat test-stimulus (HTS) -Following heat pain thresholds assessments, the thermode will be applied on the left forearm for 120 seconds at constant temperature.
The temperature will be individually adapted to induce a mean pain intensity of 60/100 based on heat pain threshold and tolerance values using the following  et al. 2013). Participant's pain intensity will be evaluated by CNPRS. If the pain intensity produced by this temperature will not induce an average pain of 60/100, the temperature will be adjusted accordingly. The 120-second HTS will be done before and after the CPT using the same thermode temperature. The thermode will be placed on adjacent areas of the forearm for every HTS trial to avoid primary skin hyperalgesia.
• Cold Pressor Test (CPT) -Participants immerse their dominant arm (up to the elbow) in circulating cold (12°C) water for 2 minutes. Participants will be instructed not to move or contract their arm during the immersion. Pain intensity ratings will be provided with the CNPRS automatically every 20 seconds during the entire 2minute immersion period.

Monitoring 24/7 physical activity
The daily physical activity will be objectively tracked by FitBit watch technology. The FitBit watch will be also wired during night for measurements of the time asleep, restless and awake, Fitbit trackers help you understand each night to make the most of each day

Exercise capacity
Participants will undergo exercise testing using a modified Balke treadmill protocol and continuous expired gas analysis. Resting and exercise vital signs will monitored continuously. The exercise duration and exercise-limiting symptoms will be recorded.
The peak VO2, VCO2 and respiratory exchange ratio (RER) will be averaged over the last 15 seconds of the exercise test. The ventilatory equivalent (VE/VCO2 slope) will be calculated from start of exercise to the end of exercise.

Mitochondrial oxygen consumption
To monitor the consumption of oxygen and extracellular acidification in intact adherent lymphocytes, Seahorse Bioscience XFe96 Extracellular Flux Analyzer will be used on the XFe96 cartridges. Further analysis of these experiments will be performed as described [12]. Metabolic profile of the individual cell lines will be generated by plotting OCR against ECAR.
Isolation of peripheral blood mononuclear cells (PBMC) will be isolated using density gradient centrifugation and will be held 3 times in 1640 RPMI medium. Cells will be frozen in freezing medium and kept in liquid nitrogen until thawed for analysis.
Total NK cell will be defined as the sum of CD56+CD16+CD3-, CD56-CD16+CD3and CD56+CD16-CD3-. The total number of NK cells in the blood will be calculated by multiplying the % cells expressing the markers above and the total lymphocyte count in the blood.

Lymphoproliferative response
Peripheral blood will be drawn at baseline and within 2 weeks after HBOT completion.
6X 10^5 PBMC will be suspended in 200 microliter RPMI 1640/10% FCS and incubated with either medium alone, 20microgram/ml PHA, PWM diluted 1:2 X10^3 to induce suboptimal lymphocyte proliferative responses or 20 U/ml IL-2. PBMC will also be incubated for 7 days with medium or with ethanol inactivated candida antigen ttrated to the lowest concentration inducing an optimal lymphocyte proliferative response. During the lat 24 h cell will be exposed to 3H-thymidine. 3H-thymidine incorporation will be measured by a direct beta-counter.
PBMCs will be ashed twice in PBS with 3% FCS. 1X10^5 cells will be resuspended in 100 microliter PBS contatining CD4,CD8, CD62, CD28, CD45, CD3conjugated colored antibodies NK cell cytotoxicity will be measured using K562 in c 51Cr release assay. PBMC (E) will be added in dif ferent concentrations to target cells (T). E/T cel ratios of 50/1, 25/1, 12.5/1 will be used. Each ratio will be incubated for 4 hours at 37 celsius. The plates will be centrifuged 10 mins, and 100microliter of supernatant will be transferred to new tubes and radioactivity will be determined. The spontateous release will be measure by incubating target cells with medium alone Maximum release will be measured by incubating target cells with 10% Triton X-100. The percentage of 51Cr release will be derermined by %Lysis = (samplespontaneous) cpm / (maximumspontaneous) cpm.

NK cytotoxicity
The cytotoxicity per NK cell will be calculated as %Lysis / (%NK cells X number of effector cells total). Index of cytotoxicity in the blood will be calculated as number of NK cells in the blood X cytotoxicity per NK cell.

Microbiome
The

Microbiome and human behavior:
The gut has been shown to have an effect on human behavior, while a broad spectrum antibiotic drug, Minocycline, has been recently suggested as a candidate treatment for depression [60] and schizophrenia [61]. Furthermore, an association between gastroenterological disease such as IBD and psychiatric morbidity (e.g. depression and anxiety) is well known, although precise mechanisms have not yet been shown. Additionally, in a human study testing effects of probiotics on brain function, women receiving the probiotics had altered brain activity in regions associated with central processing of emotion and sensation [62]. Moreover, a pilot study conducted on 39 patients suffering from chronic fatigue syndrome demonstrated a significant decrease in anxiety symptoms among patients treated with Lactobacillus casei strain Shirota for two months, compared with placebo [63].

Gut-brain signaling:
It has long been known that the brain has a strong effect on the gut. The gastrointestinal tract is sensitive to emotion in a way that anger, anxiety, sadness, and elation can all trigger gut symptoms. However, it has only recently been shown that the cross-talk between these organs is bilateral and the gut has effects on the brain as well. There appear to be different levels of signaling from the gut to the brain. One is neuronal -by bi-directional signaling, between the enteric nervous system (ENS) and the CNS via the vagus nerve. The ENS is composed of approximately 100 million nerve cells, leading to be called "our second brain" [64]. Additional indirect effects of the gut on the brain include altering function of the endocrine system. Microbes are capable of producing neurotransmitters such as norepinephrine, serotonin, and dopamine, which have effects on the CNS. Additionally, the microbiome can affect host hormone levels.
Finally, it has been shown that the microbiome can affect immune system components, altering cytokine levels and levels of inflammation.

Microbiome evaluation method:
Advances in bacterial genome sequencing is used for characterization of the human commensal bacterial community (microbiota) and its corresponding genome

Informed Consent
The investigator will obtain written informed consent from the parents of the patient participating in this study after adequate explanation of the aims, methods, objectives, and potential hazards of the study and prior to undertaking any study-related procedures. The investigator must utilize a consent form for documenting written informed consent. Informed consent will be appropriately signed and dated by the patient or the subject's legally authorized representative and the person obtaining consent.

Confidentiality
Subjects' anonymity will be strictly maintained and their identities will remain protected from unauthorized parties. The information is not to be disclosed to any third party (except for medical stuff or employees or agents directly involved in the conduct of the study or as required by law).

Study Files
The medical records will be maintained adequately to enable good data storage and latter on management. Subjects' clinical source documents will include (but not limited to) the following: subject's hospital/clinic/ hyperbaric unit records, physician's and nurse's notes, appointment book, original laboratory reports, X-ray, MRIs, and special assessment reports, consultant letters, screening and enrollment log, etc.

Randomization and handling of the control group
Since the diversity of the patients included in the study, after signing the informed consent is expected to be high, no stratifies criteria will be used. Patients will be randomized in 1:1 fashion to either HBOT or pharmaceutical group.

Adverse Events
An adverse event (AE) is any untoward medical occurrence in a clinical investigation subject administered a medicinal product and which does not necessarily have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign, symptom, or disease temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. Pre-existing events, which increase in severity or change in nature during or as a consequence of use of a medicinal product in human clinical trials, will also be considered AEs.
Any medical condition or clinically significant laboratory abnormality with an onset date before the screening visit and not related to study procedures is considered to be pre-existing, and should be documented in the case report form.

Safety Analysis Set
The safety analysis set will consist of all subjects with whom the study treatment was initiated.

Primary Efficacy Analysis Set
The primary endpoint parameter: improvement in fibromyalgia questioneers scores will be measured following the 1 st treatment period and separately after the crossed treatment period.
The primary efficacy analysis evaluation will include all subjects who completed the treatment period, had no major protocol violations.

Sample Size Considerations
According to Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT) recommendations, pain intensity reductions of 30% to 50% or more are considered useful. Therefore, the estimated sample size was calculated based on a reduction of 50% in the HBOT group and a reduction of 20% in the medication group, with a standard deviation of 40% [65,66]. Assuming a power of 80%, and 5% twosided level of significance, a total of 58 participants would be required, 29 participants in each arm. Considering a dropout rate of 15% the total sample size required is 70.